eNOS & the Radical Mechanism of Antitumor Anthracyclines

内皮型一氧化氮合酶

• 批准号: 7112219
• 负责人: BALARAMAN KALYANARAMAN
• 金额: $ 25.63万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7112219
• 关键词: SDS polyacrylamide gel electrophoresis; anthracyclines; antioxidants; apoptosis; cardiac myocytes; cardiotoxin; doxorubicin; drug adverse effect; enzyme activity; glutathione peroxidase; hydrogen peroxide; iron; laboratory rat; myocardium disorder; nitric oxide; nitric oxide synthase; northern blottings; oxidative stress; polymerase chain reaction; receptor mediated endocytosis; terminal nick end labeling; tissue /cell culture; transfection; transferrin receptor; vascular endothelium; western blottings

DESCRIPTION (provided by applicant): Long-term goal: The long-term goal of this renewal is to unravel the free radical mechanisms by which doxorubicin (DOX), a cancer chemotherapeutic drug that is currently used in the clinic, induces cardiotoxicity in cancer patients.Hypothesis: The general hypothesis to be tested is that DOX-induced apoptosis in cardiomyocytes and endothelial cells is predominantly mediated by hydrogen peroxide (H202) and that antioxidants or antioxidant enzymes that detoxify H202 are antiapoptotic. Specific Aims: First, we will investigate the effect of glutathione peroxidase (GPxI) overexpression on DOX-induced apoptosis in myocytes and endothelial cells. Next, we will demonstrate the role of endothelial nitric oxide synthase (eNOS) in DOX-induced apoptosis. The objective here is to determine whether the DOX/eNOS generated H202 is responsible for apoptosis. DOX-induced generation of reactive oxygen species (ROS) and expression of pro- and antiapoptotic proteins in myocytes and endothelial cells will be assessed using antisense eNOS. Finally, we will determine the mechanism of DOX-induced iron uptake and its implications in apoptosis. After establishing the role of ROS and eNOS in DOX-induced apoptosis, we will investigate the mechanism of antiapoptotic action of selected metalloporphyrins in this system. Methods: We will use cardiomyocytes isolated from adult rat hearts, cultured bovine aortic endothelial cells, and adenovirally transfected GPx1 overexpressing cells. Apoptosis will be detected by several methods including TUNEL analysis, caspase activity, mitochondrial cytochrome c release, Bcl-2 and Bax activity. Superoxide levels will be determined by fluorescence and spin-trapping. We will use the state-of-the-art ESR technique to detect the various redox states of metalloporphyrins. Significance: Drug toxicity is a serious side effect of cancer chemotherapy and severely limits the clinical usefulness of most widely used drugs such as doxorubicin. Children treated with DOX for leukemia develop cardiomyopathy years after cessation of DOX chemotherapy. Further understanding of the oxidative mechanisms may lead to an effective antioxidant/antiapoptotic therapy for minimizing cardiotoxicity. Novelty: Emerging literature indicate that cardiomyocyte apoptosis contributes to heart failure. Understanding the relationship between DOX-induced apoptosis and ROS formation may help discover novel antioxidant/antiapoptotic therapy in oxidant-induced disease processes.

描述（由申请人提供）：长期目标：本次更新的长期目标是阐明多柔比星（DOX）（目前临床上使用的癌症化疗药物）诱导癌症患者心脏毒性的自由基机制。假设： 待测试的一般假设是心肌细胞和内皮细胞中的DOX诱导的凋亡主要由过氧化氢（H2 O2）介导，并且使H2 O2解毒的抗氧化剂或抗氧化酶是抗凋亡的。具体目标：首先，我们将研究谷胱甘肽过氧化物酶（GPxI）过表达对DOX诱导的心肌细胞和内皮细胞凋亡的影响。接下来，我们将证明内皮型一氧化氮合酶（eNOS）在DOX诱导的细胞凋亡中的作用。本文的目的是确定DOX/eNOS产生的H2 O2是否负责细胞凋亡。将使用反义eNOS评估DOX诱导的活性氧类（ROS）的产生以及肌细胞和内皮细胞中促凋亡蛋白和抗凋亡蛋白的表达。最后，我们将确定DOX诱导的铁摄取机制及其在细胞凋亡中的意义。在确定活性氧和eNOS在阿霉素诱导的细胞凋亡中的作用后，我们将研究该系统中选定的金属卟啉的抗细胞凋亡作用机制。研究方法：我们将使用从成年大鼠心脏分离的心肌细胞、培养的牛主动脉内皮细胞和腺病毒转染的GPx 1过表达细胞。细胞凋亡将通过几种方法检测，包括TUNEL分析、半胱天冬酶活性、线粒体细胞色素c释放、Bcl-2和Bax活性。超氧化物水平将通过荧光和自旋捕获来确定。我们将使用最先进的ESR技术来检测金属卟啉的各种氧化还原状态。重要性：药物毒性是癌症化疗的严重副作用，并且严重限制了大多数广泛使用的药物如阿霉素的临床有用性。用DOX治疗白血病的儿童在停止DOX化疗数年后发生心肌病。对氧化机制的进一步了解可能会导致有效的抗氧化/抗凋亡治疗，以最大限度地减少心脏毒性。新奇：新兴文献表明心肌细胞凋亡有助于心力衰竭。了解DOX诱导的细胞凋亡和ROS形成之间的关系可能有助于发现新的抗氧化剂/抗凋亡治疗氧化剂诱导的疾病过程。

项目成果

Differences in doxorubicin-induced apoptotic signaling in adult and immature cardiomyocytes.

• DOI: 10.1016/j.freeradbiomed.2008.09.006
• 发表时间: 2008-12-15
• 期刊: FREE RADICAL BIOLOGY AND MEDICINE
• 影响因子: 7.4
• 作者: Konorev, Eugene A.;Vanamala, Sravan;Kalyanaraman, Balaraman
• 通讯作者: Kalyanaraman, Balaraman

Cytokines and lipopolysaccharides induce inducible nitric oxide synthase but not enzyme activity in adult rat cardiomyocytes.

• DOI: 10.1016/j.freeradbiomed.2008.06.017
• 发表时间: 2008-10
• 期刊: Free radical biology & medicine
• 影响因子: 7.4
• 作者: J. Vasquez-Vivar;J. Whitsett;I. Ionova;E. Konorev;J. Zielonka;B. Kalyanaraman;Yang Shi;G. Pieper

Oxidant-induced iron signaling in Doxorubicin-mediated apoptosis.

• DOI: 10.1016/s0076-6879(04)78026-x
• 发表时间: 2004
• 期刊: Methods in enzymology
• 作者: S. Kotamraju;Shasi V Kalivendi;E. Konorev;C. Chitambar;J. Joseph;B. Kalyanaraman

Synthesis and ESR studies of a novel cyclic nitrone spin trap attached to a phosphonium group-a suitable trap for mitochondria-generated ROS?

• DOI: 10.1080/10715760600911147
• 发表时间: 2007-01
• 期刊: Free Radical Research
• 影响因子: 3.3
• 作者: Yingkai Xu;B. Kalyanaraman

Nitric oxide mitigates peroxide-induced iron-signaling, oxidative damage, and apoptosis in endothelial cells: role of proteasomal function?

• DOI: 10.1016/j.abb.2003.12.037
• 发表时间: 2004-03-01
• 期刊: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
• 影响因子: 3.9
• 作者: Kotamraju, S;Tampo, Y;Kalyanaraman, B
• 通讯作者: Kalyanaraman, B